This invention relates to a method for preparing polyene salts which are water soluble compounds for use as antifungal agents. More particularly, this invention relates to a process for producing high quality antifungal products in a manner that is notable for avoiding reagents that could be very explosive and even highly toxic, and side products, avoiding over acidification, and avoiding removing aldehydes liberated during salt formation by approaches that result in emulsions which are difficult to break.
Methods already exist for preparing salts of polyene macrolide antifungal antibiotic esters. The salts of these compounds are highly water soluble and have excellent antifungal activity in vitro and in animals. For instance, U.S. Pat. No. 5,981,721 issued Nov. 9, 1999 to Mohan involved the use of diisopropylethyl amine to accomplish methylation, which results in less than optimal conversion to methyl ester and increases the side products which are over methylation products. Methods previous to Mohan are tedious, easily lead to acid degredation products, and do not apply directly to esters synthesized by methods involving a Schiff base protecting group. An older patent, U.S. Pat. No. 4,041,232, issued Aug. 9, 1977 to Sipos, et al, describes methods of converting the pure free base esters to salts, and relies upon a two-phase reaction (solid-liquid) by adding acid to the insoluble mixture in water. Undesirable side products, caused by over-acidification can result. Also, the two-phase reaction process, in most cases, failed to minimize the over acidification; and the process is slow, when minimizing the over-acidification is attempted.
Still further, the previous methods remove aldehyde liberated during salt formation by solvent extraction, but this approach results in emulsions that are difficult to break.
At pH levels less than 4.0, amphotericin B methyl ester (AME) is very unstable (Bonner, J., et al, Antibiotics, 28, 132, 1975). Another aspect to forming salts from the Schiff bases is the formation of colloids when these components are in contact with water. The colloids complicate the removal of residual aldehydes and generally result in a loss of products by these prior art methods.
Lastly, as to background, Tang U.S. Pat. No. 4,308,375, issued Dec. 29, 1981, relates generally to the same subject matter as the present invention. Nevertheless, Tang who provides a method for purifying water-insoluble polyene antibiotics, accomplishes his process, but without a Schiff base. For getting rid of gram positive and gram negative bacteria, Tang employs an ion exchange column, into which amphotericin B rich methanol is placed for the purification part of the process. The relationship with the present invention is that Tang uses an ion exchange column for purification, but otherwise presents a vastly different method unrelated to the production of a high quality antifungal, which uses tetrahydrofuran to convert a free base-Schiff base mixture to a salt form, in a single phase liquid reaction, or which uses cesium carbonate to produce a free base-Schiff base mixture. In the present invention reverse phase adsorption with an adsorption column is used to remove aldehyde. Therefore, many of the drawbacks mentioned above are likewise applicable to the Tang process.
Accordingly, a primary object of the present invention is to provide a method of methylation, which provides better conversion to the methyl ester and significantly reduces the side products which are over methylation products.
A further object of the present invention is to provide a single-phase liquid reaction to convert free base-Schiff base mixture to amphotericin B methyl ester (hereinafter AME) hydrochloride or some other salt form, thereby avoiding conversion to salts by two-phase reactions and the over acidification which results therefrom to form undesirable side products.
A still further object of the present invention is to provide reverse phase adsorption to readily remove aldehyde liberated during salt formation, instead of removing the adlehyde by solvent extraction, which results in emulsions that are difficult to break.
These and other objects of the present invention are provided in a method which features the use of cesium carbonate for converting, by methylation, to methyl ester, with methyl iodide, dimethyl sulfate or other methylating agents. To convert the free base-Schiff base mixture to AME hydrochloride, or some other salt form, the method of the present invention uses tetrahydrofuran (hereinafter THF)xe2x80x94water to dissolve the mixture for the acid treatment to obtain the salt. The THF is then removed by azeotroping, and the resulting water soluble salt of AME in water is recovered by freeze drying. The aldehyde liberated during salt formation is removed by centrifuging, which eliminates any precipitated aldehyde. The significant amount of aldehyde remaining in solution is removed by eluting the solution through the reverse phase adsorbent.
Amphotericin B is a polyene macrolide antibiotic useful in a variety of applications, such as the treatment of systemic human fungal diseases and fungal plant infections. Amphotericin B and its methyl ester, AME, have also been shown to have various attributes as an anti-protozoan, with some anti-viral activity.
In order to provide a detailed description of the present invention, in the form of a method for preparing salts of polyene macrolide esters, which utilizes cesium carbonate to reduce the amount of side products, and is more reproducible than methods of the prior art. This invention process, is now described in connection with the preparation of AME. The use of cesium carbonate as a base during the methylation part of the process, with methyl iodide, provides better conversion to the methyl ester and significantly reduces the side products which are over methylation products. Likewise, even before the previously mentioned Mohan patent, which uses diisopropylethyl amine, methylations were done with diazomethane, but this reagent is very explosive and highly toxic. A single phase liquid reation is used to convert the free base-Schiff base mixture to AME hydrochloride or some other salt form. The process used THF to dissolve the mixture for the acid treatment in order to obtain the salt. The THF is removed by azeotroping, and the resulting water soluble salt in water is recovered by freeze drying, thus avoiding the two phase reactions of the prior art, which added acid to the insoluble mixture of free base-Schiff base in water. Thereby, the tendency for over acidification is diminished, and the overall process is simplified.
In order to facilitate removal of the aldehyde protective group of the Schiff base and to form the salt of the liberated free base ester, the Schiff base is stirred in water overnight, centrifuged and the insoluble product dried by lyophilization. HPLC and 300 Mhz NMR indicate that the product contains some p-chlorobenzaldehyde and is usually about a 50/50 mixture of the desired methyl ester and the corresponding Schiff base. The mixture is stirred in ethyl acetate to remove the excess aldehyde. The product is then dissolved in a solvent which is inert to the materials, is miscible with water, and can be removed from an aqueous solution as an azeotrope. Tetrahydrofuran or acetonitrile fulfil these requirements. Solvents such as methanol which do not azeotrope with water can also be used but would require evaporation to dryness in order to remove and this would complicate redissolving. A solution of amphotericin B methyl ester Schiff base/free base in tetrahydrofuran is diluted with water to approximately 75% THF but maintained in solution. To this solution is added dilute hydrochloric acid until the appropriate pH is reached. This causes the hydrolysis of the Schiff base and forms the hydochloride salt of amphotericin B methyl ester. The tetrahydrofuran is then removed as an azeotrope with water to obtain an aqueous solution of the AME.HCl and some insoluble material containing part of the aldehyde. The residual aldehyde in the aqueous solution of the AME.HCl is an impurity which can not be readily removed by filtration, centrifuging, or solvent extraction. To remove the aldehyde, the aqueous solution of AME.HCl is passed over a reverse phase support such as Amberlite(copyright) XAD-2 (Amberlite is a registered trademark of Rohm and Haas of Philadelphia, Pa.) pre-washed with methanol and then water. The aldehyde adsorbs to the resin and essentially all the AME.HCl elutes through. The effluent from the resin is freeze-dried to obtain AME.HCl as a yellow powder which is highly soluble in water.
During the process of washing the Schiff base with water, some of the Schiff base and the liberated ester free base are dispersed in the water wash as a colloidal suspension. The aqueous colloid is readily removed from the solid Schiff base and ester free base by centrifuging. To recover product from the colloid, ammonium chloride is dissolved into the suspension at 3% weight/volume and the mixture (pHxcx9c5) is allowed to stand at 4 degrees Centigrade for 20 hours. This process converts the colloid material to AME.HCl and precipitates it from the aqueous solution. The precipitated AME.HCl is separated from the solution, dissolved in water, the residual aldehyde is removed with Amberlite(copyright) XAD-2 as described above, and the resulting aqueous solution freeze dried to obtain AME.HCl as a yellow powder.
Additionally, relating to, for instance, examples 8, 9 and 12 of the following examples, a process for preparing esters of polyene macrolide is set forth. Firstly, a mixture containing an amphoteric polyene macrolide antifungal selected from the group consisting of amphotericin B, nystatin, pimaricin, amphotericin A, rimocidin, partricin and their derivatives and anhydrous dimethylformamide is allowed to react with an aldehyde followed by the addition of cesium carbonate and methyl iodide, to produce a free base-Schiff base mixture of the methyl ester. The free base/Schiff base mixture of the ester is converted to the relatively pure free base of the ester by methods involving bisulfite or Girards T reagent and dialysis. The free base in a single phase liquid reaction is converted to amphotericin B methyl ester hydrochloride or some other salt form by using tetrahydrofuran and water to dissolve the free base for acid treatment to obtain the salt. The tetrahydrofuran is removed by azeotroping, and the water soluble salt of amphotericin B methyl ester in the water is recovered by freeze drying.
In order to illustrate the improvement enabled by the present invention, a series of laboratory procedures were carried out, beginning with the state-of-the-art method for the methylation of amphotericin B. Such examples of laboratory activity are set forth in the following examples, showing the efficacy of the present invention.